Methods for promoting growth using porcine growth hormone analogs

ABSTRACT

An analog of porcine growth hormone, is disclosed which retains the diabetogenic, insulin-sparing and lipolytic properties of porcine growth hormone while being capable of improving growth in mammals.

This is a divisional of copending application 07/920,951 filed Jul. 28,1992, now U.S. Pat. No. 5,244,822 which is a divisional of Ser. No.07/810,320 filed on Dec. 19, 1991, now abandoned which is a divisionalof 07/024,837 filed on Mar. 12, 1987, now U.S. Pat. No. 5,104,806.

BACKGROUND

The present invention relates to a class of novel analogs of porcinegrowth hormone. In particular, the present invention pertains torecombinantly-produced analogs of porcine growth hormone wherein one ormore residues corresponding to the residues at positions 32 through 38in the amino acid sequence of naturally occurring porcine growth hormoneare deleted. The present invention further relates to compositionscontaining such analogs and to the use of such analogs and compositionsto increase the growth of mammals.

The pituitary gland of normal mammals produces and secretes into thebloodstream a substance called growth hormone ("GH"). The amino acidsequences of human ("hGH"), bovine ("bGH"), and porcine ("pGH") growthhormones are similar. See Dayhoff, Atlas of Protein Sequence andStructure, Volume 5, Supplement 6, National Biomedical ResearchFoundation, Washington, 120-121 (1976); and Seeburg et al., DNA, 2,37-45 (1983). The amino acid and nucleotide sequences of salmon growthhormone ("sGH") is also known, Sekine et al., Proc. Nat'l. Acad. Sci.(USA), 82, 4306-4310 (1985). Based upon an alignment of the sequences ofbGH, hGH, ovine growth hormone ("OGH"), pGH, and sGH which provides thehighest degree of homology among these growth hormones, certain highlyconserved regions may be identified. See e.g., Dayhoff, supra, andSekine et al., supra.

In vivo, growth hormone promotes construction of protein from aminoacids, an initial fall in plasma glucose upon administration, a gradualrise in plasma glucose after the initial fall, and a breakdown of fatsinto fatty acids. The effects associated with growth horome arerespectively referred to as growth promotion (i.e., weight gain),insulin-sparing, diabetogenic and lipolytic effects. An antilipolyticeffect has also been reported, but this appears to be a facet of theinsulin-like activity of the hormone. Goodman, Metabolism, 19, 849-855(1970).

In addition, growth hormones are sililar in structure to lactogenichormones and are capable of inducing similar effects. For example, hGHdiffers from the human placental lactogen at about 15% of its residues.Wallis et al., in Growth Hormone and Related Peptides, Pecile et al.,eds., Excerpta Medica, Amsterdam, 1-13 (1976). Human growth hormonediffers from human prolactin at about 25% of its residues. Wallis etal., supra. Subcutaneous injection of bGH or recombinant bGH ("rbGH")increases milk yield in cows, goats and sheep. Eppaird et al., J. DairySci, 68, 1109-1115 (1985); Bauman et al., J. Dairy Sci., 68, 1352-1362(1985); Hart, Proc. Nutr. Soc., 42, 181-194 (1983); and see Hart et al.,Biochem. J., 218, 573-581 (1984).

The isolation of growth hormone from pituitaries involves lysingpituitary cells associated with production of the hormone. However, thelysing of cells releases proteolytic enzymes (proteases) which maycleave at least some of a naturally-occurring pituitary growth hormone("nGH") into fragments. Futhermore, once secreted into the bloodstream,nGH is exposed to proteases which may cleave nGH into the same or intodifferent fragments. A major area of investigation for growth hormonefragment research is directed at a determination of whether nGH or itsfragments or both give rise to the actions associated with growthhormones which have been extracted or which are circulating in thebloodstream. In this regard, it may be noted analogs of hGH renderedresistant to digestion by the protease trypsin by chemically modifyinglysine or arginine residues possess significant, albeit attenuated,growth-promoting, diabetogenic and insulin-like activities. Cameron etal., Biochim. Biophys. Acta, 254-260 (1985). Nevertheless, discreteportions ("domains") of the nGH molecule are believed to be responsiblefor one or another of the effects of nGH. To the extent thatresponsibility for the actions of nGH may be localized in this way,fragments and analogs may be produced in which the protein-synthetic,insulin-sparing, diabetogenic and lipolytic effects are selectivelyaltered.

As used hereinafter, the positions of amino acid residues present infragments or analogs of growth hormone are identified in a subscriptwherein numbers indicate the presence of the residues found at the samepositions in the corresponding nGH and wherein deletions are indicatedby a comma. For example, naturally occurring porcine growth hormone isrepresented by pGH₁₋₁₉₀.

A 20,000-dalton variant ("20K") of hGH (22,000-dalton) which may beisolated from pituitaries and which corresponds to hGH₁₋₃₁,47-191promotes growth in hypophysectomized rats, is not hyperglycemic orhyperinsulinemic in dogs, is neither insulin-sparing nor lipolytic invivo or in vitro, and is less reactive in radioimmunoassays for hGH thanis hGH itself. Lewis et al., J. Biol. Chem., 253, 2679-2687 (1978);Frigeri et al., Biochem. Biophys. Res. Commun., 91, 778-782 (1979);Lewis et al., Biochem. Biophys. Res. Commun., 92, 511-516 (1980); andLewis et al., Endocr. Res. Commun., 8, 155-164 (1981). The 20K variantof hGH is a product of post-transcriptional modification. Lewis et al.,Biochem. Biophys. Res. Commun., supra. It may be the case that the 20Kvariant of hGH may be a more important growth promoter than would bepredicted from its in vitro bioactivity due to its tendency to dimerizeand thus escape renal degradation. Baumann et al., Endocrinology, 117,1309-1313 ( 1985).

Fragments of hGH which include residues deleted from 20K hGH have alsobeen disclosed. Although none of these fragments are reported to promotegrowth, some exhibit properties of potential relevance to thediabetogenic and lipolytic properties of growth hormone.

A synthetic fragment corresponding to residues 31-44 of hGH is lipolyticin vivo in starved animals and in vitro [Yudaev, et al., Biokhimiya, 41,843-846 (1976)] but stimulates glucose uptake (i.e. was insulin-sparing)only after in vitro preincubation in the absence of GH, anon-physiological state. Yudaev, et al., Biochem. Biophys. Res. Commun.,110, 866-872 (1983). Some peptides analogs of hGH are diabetogenic butan analog of hGH₅₂₋₇₇ is not. Lostroh, et al., Diabetes, 27, 597-598(1978). A peptide consisting of hGH₂₀₋₄₁ is devoid of activity. Reagan,Diabetes, 27, 883-888 (1978). A peptide consisting of hGH₁₋₃₆ is devoidof effect on blood glucose or on growth. Chillemi, et al., in GrowthHormone and Related Peptides, Pecile, et al., eds., Excerpta Medica,Amsterdam, 50-63, (1976).

However, a peptide corresponding to hGH₃₂₋₄₆ causes a decrease in serumfree fatty acids, and is insulin-sparing when coadministered withinsulin in vitro [Frigeri et al., in Proceedings, 64th Annual Meeting ofthe Endocrine Society, San Francisco, 101 (Abstract 88) (1982)] and invivo [Rudman, U.S. Pat. No. 4,558,033, and Stevenson et al., Diabetes,33, 149A (Abstract No. 572) (1984)]. Fragments and analogs (involvingsubstitution of heterologous amino acids or stereoisomers) of hGH₃₂₋₄₆are also insulin-sparing when coadministered with insulin in vivo. Joneset al., copending and coassigned U.S. patent application Ser. No.501,024.

SUMMARY OF THE INVENTION

The present invention relates to a class of porcine growth hormoneanalogs which retains the biological activity and properties ofnaturally occuring porcine growth hormone while increasing the growthrate, feed efficiency, increased lypolysis or milk yields.

In particular, the present invention relates to a recombinant porcinegrowth hormone analog comprising the amino acid sequence

    Z-pGH.sub.1-31 -(X).sub.n -pGH.sub.39-190

wherein

n is 0 or 1;

Z is hydrogen, MET, ALA or MET-ALA-; and

X is a peptide of an amino acid residue(s) comprising

    -GLU-ARG-ALA-TYR-ILE-PRO-GLU-

wherein one or more of the amino acids are deleted; and allelic versionsthereof.

The invention further relates to a synthetic gene encoding porcinegrowth hormone analogs having the above described sequence. The presentinvention also relates to processes of construction of variousreplicable cloning vehicles harboring the DNA sequences as well asexpression vehicles harboring DNA sequences useful to direct theproduction of the porcine growth hormones analogs in transformedbacteria or transfected cell lines. In addition, the present inventionprovides for a gene encoding the analogs of porcine growth hormonehaving the above-described amino acid sequence. The present inventionalso encompasses the various replicable cloning vehicles, expressionvehicles, and transformed bacterial or cell cultures, all harboring thealtered genetic information necessary to effect the production of theporcine growth hormone analogs of the present invention.

The porcine growth hormone analogs of the present invention are producedin substantially pure form and therefore exist essentially free of otherproteins of porcine origin. The porcine growth hormone analogs may beformulated with conventional suitable carriers and adjuvants, includingother proteins, for example, serum albumin, to yield acceptablecompositions so as to facilitate efficacious delivery to a host animal.

The present invention also provides a method for promoting growth in ananimal which involves administering to an animal an effective dose of aporcine growth hormone analog of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of the pBR 322-Trp-pGH plasmidconstruction;

FIG. 2 is a schematic illustration of the pCFM 414-Trp-pGH plasmidconstruction;

FIG. 3 is a diagrammatic description of the oligonucleotide assemblyutilized to make the XbaI to ApaI pGH DNA fragments utilized in theconstruction of pGH and pGH-7; and

FIG. 4 is a diagrammatic illustration of the components utilized in theconstruction of pCFM 846-pGH plasmid.

DETAILED DESCRIPTION

As previously discussed, the physiological activities of growth hormonesmay be attributed to the different domains of the intact polypeptide.Such activities may also be due to a particular folding or modificationof the intact polypeptide, to the release of mediating factors, or to"contamination" by other pituitary peptides, e.g. α- and β-lipotropinwhich themselves can be responsible for lipolytic activity [Kuhn et al.,J. Clin. Endocrinol. Metab., 56, 1338-1340 (1983)]. Frigeri et al.,Hormone Res., 17, 197-201 (1983).

One way to separate the effects of contaminants from the effects ofpurified hormones is to examine the activities of a growth hormone whichis produced in isolation from other pituitary components, e.g.recombinant pGH ("rpGH"). The gene for pGH has been sequenced and hasbeen expressed in prokaryotic and eukaryotic cells in a variety offorms. Keshet et al., Nucleic Acids Res., 9, 19-30 (1981); Woychik etal., Nucleic Acids Res., 10, 7197-7210 (1982); Seeburg et. al., DNA, 2,37-45 (1983); Kopchick et al., DNA, 4, 23-31 (1985); and George et al.,DNA, 4, 273-281 (1985).

The present invention provides purified and isolated polypeptideproducts having one or more of the biological properties (e.g.,immunological properties and in vitro biological activity) and physicalproperties (e.g., molecular weight) of naturally-occurring pGH. Thesepolypeptides are also characterized by being the product of chemicalsynthetic procedures or of prokaryotic or eukaryotic host expression(e.g., by bacterial, yeast, higher plant, insect and mammalian cells inculture) of exogenous DNA sequences obtained by genomic cloning by cDNAcloning or by gene synthesis. The products of typical yeast (e.g.,Saccharomyces cerevisiae) or prokaryote (e.g., Escherichia coli (E.coli) host cells are free of association with any mammalian proteins.The products of microbial expression in vertebrate (e.g., non-humanmammalian and avian) cells are free of association with any humanproteins. Depending upon the host employed, polypeptides of theinvention may be glycosylated with mammalian or other eukaryoticcarbohydrates or may be non-glycosylated. Polypeptides of the inventionmay also include an initial methionine amino acid residue (at position-1).

As used herein, the term "peptide of amino acid residues" refers topeptide comprising the amino acids GLU-ARG-ALA-TYR-ILE-PRO-GLU whereinone or more amino acids have been deleted. For the purposes of thepresent invention, the deletion of the amino acids in the peptide thusdescribed may be sequential or random.

The term "manufactured" as applied to a DNA sequence or gene shalldesignate a product either totally chemically synthesized by assembly ofnucleotide bases or derived synthesized. As such, the term is exclusiveof products "synthesized" by cDNA methods of genomic cloningmethodologies which involve starting materials which are initially ofbiological origin.

As used herein, the term "allelic versions" refers to modifications ofone or more amino acids in the sequence of the porcine growth hormoneanalogs of the present invention without altering the biologicalactivity of the analog. Such allelic versions are readily ascertained byone of ordinary skill in the art.

It should be noted that if Z is MET-ALA, preferably the MET residue isprocessed off to yield an analog wherein Z is ALA. A preferred porcinegrowth hormone analog of the present invention comprises a porcinegrowth hormone analog of formula (I) wherein n is 0 and Z is ALA.Another preferred porcine growth hormone analog comprises a porcinegrowth hormone analog of formula (I) wherein n is 1, Z is ALA and X is aresidue having the sequence: -GLU-ARG-ALA-GLU- ("ALA-pGH₁₋₃₄,38-190 ").

A further preferred porcine growth homone analog of the presentinvention comprises an analog of formula (I) wherein n is 1, Z is ALAand X a residue having the sequence: GLU-ARG-ALA-TYR-ILE-GLU("ALA-pGH₁₋₃₆,38-190 "). An additionally preferred porcine growthhormone analog of the present invention comprises an analog of formula(I) wherein n is l, Z is ALA and X is a residue having the sequence:-GLU-ALA-TYR-ILE-PRO-GLU-("ALA-pGH₁₋₃₂,34-190 ").

Table 1 represents the amino acid sequence of naturally occurring pGH.

    TABLE 1      ##STR1##      ##STR2##      ##STR3##      ##STR4##      ##STR5##      ##STR6##      ##STR7##      ##STR8##      ##STR9##

The compositions and methods of the present invention utilize aneffective amount or dose of the porcine growth hormone analogs of thepresent invention. As used herein, the term "effective amount or dose"of the porcine growth hormone analog refers to an amount of porcinegrowth hormone to be administered to an animal in order to produce anincrease in growth or related properties, i.e., feed efficiency, leanercarcass composition, milk production, etc. Such effective amounts ordoses are readily ascertained by one of ordinary skill in the art.

The following examples serve to further illustrate the embodiments ofthe present invention.

EXAMPLE 1

Sixty micrograms of polyadenylated RNA was isolated from one gram ofporcine pituitaries. cDNA was generated from the polyA RNA via theprocedures described by Okayama et al., Mol. Cell. Biol., 2, 161 (1982)and transformed into an competent E. coli (strain HB101). Five thousandcolonies were screened using a ³² p-labeled nick-translated 493 bp PvuIIcDNA bGH probe. Two hundred colonies hybridized from which five werecarried through secondary screening, DNA isolation and restrictionenzyme mapping. Two isolates contained approximately 900 bp of pGH cDNAsequence (1,4) and three contained approximately 700 bp (2,3,5).

Clone number 1 was carried through M13 DNA sequencing then transferredinto a pBR322-Trp expression vector. To achieve this construction,plasmid DNA from clone 1 was cut at a HaeII site near the 5' end of theinsert and at a HaeII outside the 3' end of the cDNA insert in thepBR322 region of clone 1, generating an 1100 bp fragment. This 1100 bpsegment was SI nuclease treated and XbaI-Blunt linker was ligated toboth ends. The XbaI bracketed DNA segment was then ligated into apBR322-Trp expression plasmid that had been cut with XbaI (FIG. 1).

The XbaI-Blunt linker supplied a 5' XbaI site and 3' blunt end, as wellas an ATG initiation codon. ##STR10##

The polyA tail at the 3' end was removed by carrying the aboveexpression plasmid through a PvuII and EcoRI digestion to release theTrp/pGH 355 bp fragment. To this, a PvuII-BamHI linker was ligated atthe PvuII cut 3' end and this was then ligated into a pCFM 414expression plasmid that had been cut with the restriction enzymes EcoRland BamHI (FIG. 2).

The PvuII-BamHI linker contained one half of the PvuII site, atermination codons (TAA) and a BamHI site. ##STR11##

Based upon the sequencing data, two different double stranded (ds)oligonucleotide sequences were chemically synthesized as described above(PGHwt, PGH-7). Each of these DNAs was assigned to have an Xbalrestriction site at the 5' end and an ApaI restriction site at the 3'end. Just downstream of the ApaI site a HindIII restriction site wasadded to facilitate cloning into m13mp19 bacteriophage.

Briefly stated, the protocol employed in the following examples toconstruct a manufactured gene was generally as set out in the disclosureof co-owned Alton, et al., PCT Publication No. WO83/04053, which isincorporated by reference herein. The genes were designed for initialassembly of component oligonucleotides into multiple duplexes which, inturn, were assembled into discrete sections. These sections weredesigned for ready amplification and upon removal from the amplificationsystem, could be assembled sequentially or through a multiple fragmentligation in a suitable expression vector.

EXAMPLE 2

Two gene fragments for porcine growth hormone were constructed inparallel. One gene fragment (Fragment 1-pGHwt) contained a 22K porcinegrowth hormone gene sequence as deduced by cDNA sequencing. A secondgene fragment (Fragment 2-pGH-7) coded for the 22K porcine growthhormone sequence minus 7 amino acids in the "deletion peptide" (DP)region (hereinafter 21K porcine growth hormone). Oligomers 15, 16, 17and 18 bridged the gap between the beginning and the end of the DPcoding region in pGH-7 and code for the amino acids added in this geneversion. Oligonucleotide segments common to both gene versions were 1,2, 3, 5, 6, 7, 8, 9, 10, 12, 13, 14, 16 and 18 (FIG. 3).

The 20 oligonucleotides required for the construction of the two genefragments were synthesized on an ABI DNA synthesizer and purified by gelelectrophoresis using standard methods and are listed in Table 2. Eachpurified oligonucleotide (oligo) was dissolved in 1 ml of TE (10 mM TrisHCl, pH 7.2, 0.1 mM EDTA) and the absorbance at 260 nm was recorded. Theabsorbance was compared to the calculated extinction coefficient for theoligo and a concentration was computed. The concentration in picomolesper microliter was then used to measure the respective oligos for thegene constructions. For example, for oligo #1, a 39-mer containing 13adenines, 8 cytosines, 9 guanines and 9 thymidines, the calculatedextinction coefficient at 260 nm is 444700. The absorbance at 260 nm wasmeasured as 0.379 and the resulting concentration in pm/microliter is0.852. The oligos were measured using an Eppendorf pipetman intoEppendorf tubes. The amounts used were such that 100 picomoles of eacholigo would be allotted to each gene fragment to be constructed. Thus,200 pmole of oligos #1, 2, 3, 5, 6, 7, 8, 9, 10, 12, 13, 14, 16 and 18were used since these are common to each of the two gene constructions.100 pmole of oligos 15, 17, 19 and 20 were used since they are found inonly one of the gene constructions. The measured oligos were speed-vac'dto dryness and then redried using 150 microliters of 80% ethanol, 20%water.

The oligos were selectively phosphorylated so that the oligomers whichwould ultimately be on the ends of the finished gene constructions couldnot self-ligate. Thus oligos 1 and 14 were not phosphorylated. All theother oligos were phosphorylated. All kinations and ligations were donein ligation buffer, "LB", consisting of 50 mM HEPES, pH 7.6, 10 mMmagnesium chloride and 10 mM dithiothreitol. (LB was stored as aten-fold concentrated solution, 10X LB and diluted into water asneeded.) Oligos 1 and 14 were each dissolved in 60 microliters of LB andset on ice until needed later.

A kinase mix was prepared which contained: 92 microliters 10X LB, 20microliters polynucleotide kinase (Boehringer-Mannheim, 10unit/microliter), 1 microliter 10 mM ATP, in TE, 1/4 microliter32-phosphorus gamma, phosphate ATP containing 80,000,000 counts/minuteof radioactivity, 810 microliters water.

The total volume of the kinase mix was 920 microliters. The mix wasadded to the dried oligos, 20 microliters for each 100 pmole to bephosphorylated. Oligos 2, 3, 5, 6, 7, 8, 9, 10, 12, 13, 16 and 18 (200pmole each) were each dissolved in 40 microliters of kinase mix. Oligos15, 17, 19 and 20 (100 pmole each) were dissolved in 20 microliters ofkinase mix. All the tubes containing the dissolved oligos in kinase mixwere then incubated at 37° C. for 45 minutes. A 1/4 microliter aliquotwas removed from each tube and spotted separately on a DE-81 paperstrip. the DE-81 strips were then eluted in a chromatography chamberwith 0.35M ammonium formate buffer until the solvent front reached thebottom of the descending strip. The strips were then removed from thechamber, dried in a 100° C. drying oven and cut into pieces for analysisin a liquid scintillation counter (Beckman LS 6800). The strips were cutsuch there was one piece containing only the origin and a second piecewhich contained the rest of the DE-81 strip. The fragments from theDE-81 strip were then placed in plastic counting vials and counted dryin the LS-6800. The counter showed radioactivity incorporated at theorigin of each strip and so the phosphorylation reactions were chasedwith a large excess of cold ATP. One microliter of 10 mM ATP was addedto each phosphorylation reaction and the tubes were incubated at 37° foran additional 45 minutes following the DE-81 analysis. All oligocontaining tubes (including 1 and 14 which were not phosphorylated) werethen boiled for 5 minutes and quickly cooled. This process destroyed thekinase enzyme.

    1+8

    2+9

    3+10

    5+12

    6+13

    7+14

    15+17

    16+18

    19+20

Each duplex took the name of the first oligo in the pair. Tubes 1, 2, 3,5, 6, 7, 15, 16 and 19 were mixed, boiled for 5 min. and then slowcooled to room temperature.

These annealed duplexes were then combined to form tetramers:

    3+2

    5+6

These tetramers took the name of the first tube in the pair, 3 and 5,respectively. To each of tubes containing tetramers 3 and 5 were added 5microliters of 10 mM ATP (to give approximately 200 micromolar ATPconcentration). The tetramers were annealed at 37° for 10 minutes. Fivemicroliters of ligase (Boehringer-Mannheim, 1 unit/microliter) wereadded to each tube. The ligations were incubated for 5 minutes at 37°and then for 1 hour on ice.

One-half of duplex 16 was combined with duplex 15 and the remaining 1/2with duplex 19. To each of these tetramers were added 2 microliters of10 mM ATP. The tetramers were annealed at 37° for 10 minutes, then 2microliters of ligase were added to each tube. The mixtures wereincubated 5 minutes at 37° and for one hour on ice.

More duplexes were added to yield longer duplexes: duplex 7 added totetramer 5, yielding hexamer #5; duplex 1 added to tetramer 3, yieldinghexamer #3; 2.5 microliters 10 mM ATP and 2.5 microliters ligase wereadded to these hexamers. The two tubes were then mixed, incubated for 10minutes at 37° and for one hour on ice.

One-third of hexamer #5 was added to each of tubes 15 and 19 containingthe central oligos of pGH-7 and pGH, respectively. The tubes 15 and 19were incubated 10 minutes at 37° and 2 hours on ice. Then hexamer #3 wasadded, 1/2 to each of tubes 15 and 19. All oligos required forconstruction of 10 mM ATP and 5 microliters of ligase were added to eachof the tubes. The tubes containing the ligation mix were incubated for10 minutes at 37° and at 4° for 5 days.

Five microliters from each of the three ligations were checked on a 0.75mm thick analytical polyacrylamide gel, 5%, 7M urea. 32-P labeled HpaIIcut pBR322 was run in an adjacent lane as a standard. The aliquots fromeach ligation as well as the HpaII standard were diluted into 20microliters of 80% formamide 20% water containing 0.1% xylene cyanol andbromphenol blue dyes (80% formamide plus dyes). The samples were boiledfor 5 minutes then quick chilled on ice and loaded onto the gel. The gelwas run at 400 volts for 45 minutes (until the bromphenol blue reachedthe bottom of the gel slab) then removed from the glass plates, wrappedin Saran Wrap and placed in a film cassette with a sheet of DupontCronex X-ray film. Exposure at -70° C. and developing of the film causedthe ligated DNA to be visualized on the autoradiogram. In lane 15 was aband corresponding to a 272 base pair duplex. In lane 19 was a bandcorresponding to a 293 base pair duplex. This indicated that eachligation had proceeded to give some of the desired DNA construct forpGH-7 and pGH, respectively.

Based on the analytical gel results, the three ligation mixtures wereprepared for large scale gel purification. Each ligation was ethanolprecipitated using 40 microliters of 3M sodium acetate and 1 ml of 100%ethanol and chilling at -70° C. overnight. After a 10 minutecentrifugation at 10,000 times g, the DNA pellet was isolated byremoving the supernatant and rinsing with 100 microliters of ice cold80% ethanol. The rinsed pellets were speed-vac'd to dryness. Each pelletwas then dissolved in 80 microliters of 80% formamide with dyes. Theywere boiled for 5 minutes and quick chilled on ice. 1/2 of each ligationmixture was then loaded onto a 3 mm thick 5% polyacrylamide gelcontaining 7M urea. The gel was run at 250 to 300 volts for 2 hours. Thegel was then placed into a film cassette and autoradiographed at roomtemperature to visualize the bands. The developed film was then placednext to the gel in the cassette, the position of the band in each lanecorresponding to the fully ligated gene fragment was marked and thedesired bands were cut out of the gel using a razor blade. Each gelslice was then pulverized by extruding it through a 3 ml syringe(without needle) into an Eppendorf tube. Each pulverized gel slice wascovered with 0.7 ml of gel elution buffer (0.5M ammonium acetate, 0.01Mmagnesium acetate, 0.001M EDTA and 0.1% ammonium acetate, 0.01Mmagnesium acetate, 0.001 M EDTA and 0.1% sodium dodecyl sulfate) andincubated overnight at 37° C.

The gel-solution mixture was filtered through a glass fiber filterpad ina syringe barrel and washed 3 times with n-butanol. The DNA wasprecipitated by addition of 2 1/2 volumes of ethanol and storage at -70°for 1 hour. Centrifugation for 10 minutes at 1000 times g gave pelletswhich were isolated by decanting the supernatant. The pellets werespeedvac'd for 5 minutes to dry them. They were redissolved in 200microliters of TE, centrifuged to concentrate the polyacrylamide residueand then reprecipitated with 20 microliters 3M sodium acetate and 550microliters of 100% ethanol. The dried DNA pellets were counted on theliquid scintillation counter and found to contain about 1 picomole ofeach gene fragment.

Each DNA pellet was redissolved in 20 microliters of solution containing2 microliters of 10X LB and 1 microliter of radiolabeled ATP (30,000,000cpm per microliter). One-fourth microliter aliquots were removed fromeach tube and spotted onto DE-81 strips. One microliter ofpolynucleotide kinase was added to each tube and the tubes wereincubated at 37° for 30 minutes. One-fourth microliter aliquots wereremoved from the phosphorylation reactions and spotted onto another setof DE-81 strips. All six strips were then eluted with 0.35M ammoniumformate and their origins compared for retained radioactivity.Comparison of the before and after DE-81 strips for each duplex showedthat phosphorylation had occurred so each reaction was chased with 1microliter 10 mM ATP to complete the reaction. After 45 minutesincubation at 37°, the tubes were boiled for 5 minutes and allowed toslow cool to room temperature. The annealed phosphorylated duplexes inligation buffer were ready for ligation into the appropriately cloningvectors.

                                      TABLE 2                                     __________________________________________________________________________    1 5'-                                                                             CTAGAAGGAGGAATAACATATGGCTTTTCCAGCAATGCC - 3'                              2 5'-                                                                             TCTCTCGAGCCTGTTCGCTAACGCTGTACTGCGTGCTCAG - 3'                             3 5'-                                                                             CACCTGCATCAACTGGCTGCAGACACTTACAAAGA - 3'                                  5 5'-                                                                             TCCCGGCGCCAACTGGTAAAGACGAAGCTCAACA - 3'                                   6 5'-                                                                             GAGATCTGATGTTGAACTGCTGCGTTTCTCT - 3'                                      7 5'-                                                                             CTGCTGCTGATTCAATCTTGGCTGGGGCCCTTCA - 3'                                   8 5'-                                                                             GAGAGGCATTGCTGGAAAAGCCATATGTTATTCCTCCTT - 3'                              9 5'-                                                                             AGGTGCTGAGCACGCAGTACAGCGTTAGCGAACAGGCTCGA - 3'                            10                                                                              5'-                                                                             GAATTCTTTGTAAGTGTCTGCAGCCAGTTGATGC - 3'                                   12                                                                              5'-                                                                             TCTCTGTTGAGCTTCGTCTTTACCAGTTGGCGCC - 3'                                   13                                                                              5'-                                                                             GCAGAGAGAAACGCAGCAGTTCAACATCAGA - 3'                                      14                                                                              5'-                                                                             AGCTTGAAGGGCCCCAGCCAAGATTGAATCAGCA - 3'                                   15                                                                              5'-                                                                             ATTCGGTCAGCGTTACTCTATC - 3'                                               16                                                                              5'                                                                              CAGAACGCTCAGGCTGCATTTTGCTTCTCTGAAACCA - 3'                                17                                                                              5'                                                                              TTCTGGATAGAGTAACGCTGACC - 3'                                              18                                                                              5'                                                                              GGGATGGTTTCAGAGAAGCAAAATGCAGCCTGAGCG - 3'                                 19                                                                              5'                                                                              ATTCGAACGTGCTTACATCCCGGAAGGTCAGCGTTACTCTATC - 3'                          20                                                                              5'                                                                              TTCTGGATAGAGTAACGCTGACCTTCCGGGATGTAAGCACGTTC - 3'                         __________________________________________________________________________

After cloning each of the synthetic pGH constructions into M13mp19,single stranded (ss) and ds DNAs were isolated. The DNAs were sequenced.The ds DNAs were used as sources of an XbaI to ApaI ds DNA fragmentscontaining the 5' portion of the three different pGH constructions.

To construct the pGH expression vectors three way DNA ligations wererequired (FIG. 4). Component 1 was the XbaI to ApaI ds DNA fragmentisolated from one of the three pGHmp19 DNAs. Component 2 was isolatedform a pCFM414pGH vector as an ApaI to BamHI ds DNA fragment containingthe 3' of the pGHwt gene. Component 3 was a pCFM846 plasmid cut withXbaI and BamHI. The pCFM846 plasmid is a derivative of pCFM836(described below) prepared by inserting the following DNA sequencebetween the unique ClaI and KpnI site of pCFM836: ##STR12## The plasmidpCFM836 is prepared as a derivative of pCFM536 (ATCC#39934) constructedto incorporate a Kanamycin resistance marker, a synthetic P1 promoter, anew cloning cluster of restriction sites, and a series of translationalstop sequences designed to stop translation in all three reading frames.The β-lactamase gene is first deleted by digestion of pCFM536 with SstIand XbaI. This serves to delete not only the marker gene but also theentire "par" or stability sequence, the P1 promoter, and part of thecluster of restriction sites. The Kanamycin gene sequence may beobtained as a SmaI to HindIII fragment from the Tn5 plasmid of Beck etal., Cold Spring Harbor Symp. Quant. Biol, 45, pp. 107-113 (1981). Toprepare the fragment for insertion into the new vector, a SstI linker isadded to the SmaI site and an NdeI linker added to the HindIII site. Thenaturally occurring NcoI restriction site in the Kanamycin resistancegene was destroyed by site specific mutagenesis at the codon for athreonine residue 76 amino acids upstream of the carboxy terminalleucine specified by the Kanamycin resistance gene and specifically byaltering the ACC codon to an ACT codon. The "par" locus sequence may beobtained as a HincII to AvaI digestion fragment of pSC101 (ATCC#37032).To prepare the "par" fragment for insertion into the new vector, theHincII is first treated with a SalI linker and then an AatII linker. TheAvaI site is treated with a BamHI linker and then an NdeI linker. A DNAsequence containing a synthetic P_(L) promoter obtained by chemicalsynthesis of a ds DNA oligonucleotide with sticky ends for insertionbetween an AatII restriction site and an XbaI restriction site was addedas follows: ##STR13## To add the translation stops in all three framesthe plasmid was cut with BamHI and the following ds oligonucleotideinserted: ##STR14##

After ligation each of the constructions was transformed into E. coli(strain FM6). FM6 is a derivative of AM7 (#CG608159) that has beenrendered phage resistant to several unknown bacteriophages and containsthe gene encoding tetracycline resistance and the lambda bacteriophagerepressor genes, CI857 and cro, integrated into the chromosome.

After transformation, representative clones for each type of pGH werepicked. Covalently closed circular (ccc) plasmid DNA was isolated andsequence using the alkaline denatured ccc technique. The sequence of thesynthetic portion of 21K pGH is given in Table 3.

                                      TABLE 3                                     __________________________________________________________________________     ##STR15##                                                                    TTCCTCCTTATTGTATACCGAAAAGGTCGTTACGGAGAGAGC                                     ##STR16##                                                                    TCGGACAAGCGATTGCGACATGACGCACGAGTCGTGGACGTAGTTGACCGACGTCTGTGA                   ##STR17##                                                                    ATGTTTCTTAAGCCAGTCGCAATGAGATAGGTCTTGCGAGTCCGACGTAAAACGAAGAGA                   ##STR18##                                                                    CTTTGGTAGGGCCGCGGTTGACCATTTCTGCTTCGAGTTGTCTCTAGACTACAACTTGAC                   ##STR19##                                                                    GACGCAAAGAGAGACGACGACTAAGTTAGAACCGACCCCGGGAAGTTCGA                            __________________________________________________________________________

EXAMPLE 3

In addition, four additional pGH analogs may be constructed by sitedirected mutagenesis. First, the small XbaI to BamHI fragment fromp846pGH22K is cloned into M13mp10 and single stranded phage DNA isisolated. Primers for each of the analogs are synthesized and kinasedand annealed to the single stranded DNA. The four dNTPs, ATP, T₄ DNAligase and the klenow enzyme are added to synthesize the second strandof the DNA containing the desired changes. The DNA is transfected intothe host strain, JM103 and plaques are plated out. The correct clonesare determined by hybridization to ³² P-labeled primers of the analogsequence. Sequences are confirmed by dideoxy sequencing and the XbaI toBamHl fragment from M13mp10 is cloned back into p846. The primers forthe clones are listed below:

Analog 1: (Tyr, Ile, Pro) TCGAACGTGCTGAAGGTCAGCG

Analog 2: (Pro) GTGCTTACATCGAAGGTCAGCG

Analog 3: (Arg) CAAAGAATTCGAAGCTTACATCCC

The fourth clone (-Glu, Arg . . . Glu-) requires two sequential sitedirected mutageneses. The first primer (A) will remove the Glu, Arg.After this site directed mutagenesis, the single stranded DNA ispurified and a second round of site directed mutagenesis is conductedwith a second primer (B) which will remove the second Glu.

Analog 4 Primers: ##STR20##

EXAMPLE 4

A fermentation run was performed for each of 22K and 21K. Cell densitieswere reached up to an optical density of 65. The amount of porcinegrowth hormone or analog thereof formed varied from 50-75 mg/OD L (3-3.9gm/L).

The runs were made using E. coli FM6/pCFM 856 pGH #3 (with 22K pGH) and#8 (with 21K pGH). Each of the above have a walkaway plasmid and akanamycin drug marker for plasmid stability.

                  TABLE 4                                                         ______________________________________                                        (Medium composition)                                                          Ingredient        Batch (8L)      Feed 3L                                     ______________________________________                                        yeast extract     40    gm        400  gm                                     (NH.sub.4).sub.2 SO.sub.4                                                                       30    gm        15   gm                                     K.sub.2 HPO.sub.4 56    gm        --                                          KH.sub.2 PO.sub.4 64    gm        --                                          Dow P-2000        2     ml        --                                          Glucose           40    gm        1300 gm                                     (MgSO.sub.4 --7H.sub.2 O) (1M)                                                                  32    ml        103  ml                                     Trace Metal Solution                                                                            16    ml        28   ml                                     Vitamin Solution  16    ml        28   ml                                     Kanamycin         20    μg/ml                                              ______________________________________                                    

The fermentation runs were made in a batch-fed mode and under carbonlimitation. A temperature of 30° C. and a pH of 7.0 were maintained.Dissolved oxygen was maintained at 50% air saturation. Samples weretaken at regular intervals and growth and acetate levels were measured.pGH concentration was measured using Coomassie stained SDS-PAGE. Becauseof the lack of availability of pure pGH, pure bGH and IFN- αCon wereused as a standard. The gel was scanned using a Shimodzuintegrator/scanner and the average of the two standards was used tocalculate the concentration of pGH and percentage of total protein.

Cells were induced by raising the temperature to 42° C. at an OD of ˜25and were observed under a microscope for inclusion bodies. It contained1-2 inclusion bodies after about 3-4 hr of induction. There was no pGHfound in pre-induction samples from two analog runs. The results areshown in Tables 5 and 6 for wild type (22K) and analog (21K).

                  TABLE 5                                                         ______________________________________                                        Time of                                                                       Fermentation         Percentage of                                            (hr)          OD     Total Protein                                                                             mg pGH/OD L                                  ______________________________________                                        Gel results for wild type pGH (22K)                                           Start  18.5       25.4   Pre-induction 2                                                                          6-10                                      Induction                                                                            21.0       43.0   10-15     30                                                23.0       47.0   20-25     35-40                                             25.5       48.5   30        65                                                27.5       52.7   30-35     70-75                                      Gel results for Analog pGH (21K)                                              Start  18.5       24.8   Pre-induction 0                                                                         0 - negl.                                  Induction                                                                            21.0       48.0   20        45                                                23.0       63.0   25-30     65                                                25.5       57.2   30-35     65-70                                             27.5       58.3   30-35     60-65                                      ______________________________________                                    

As illustrated in these tables, a maximum amount of pGH (70-75 mg/OD L)was produced with the 22K pGH. With the 21K pGH analog it seems that theamount of analog produced peaked with OD and later with a decrease in ODit started going down, possibly due to proteolytic degradation. It showsthat 5-6 hours after induction is probably the optimum time to stopfermentation.

EXAMPLE 5

The biological activity of recombinantly-produced analogs evaluated inhypophysectomized rats during a 10-day weight gain bioassay, usingpituitary-derived porcine growth hormone as a standard.

The 21K pGH analog of the present invention and pituitary-derived pGH("pd-pGH") preparations were injected at three doses each, twice dailyin 0.1 ml volumes, by subcutaneous route to hypophysectomized femalerats on days 0-9 following a 12-day acclimation period. The pd-pGHpreparation was reconstituted from lyophilized form in pH 9.5bicarbonate buffer (30 mM) to produce a stock solution having aconcentration of 1 mg/ml. The stock solution was diluted, as were allrecombinant samples, in "World Health Organization" ("WHO" buffer (0.2%lactose, 0.2% mannitol, 30 mM NaHCO₃, pH 8.6) to 300, 100, and 30 μg/ml.The samples were stored at 4° C. for the duration of the experiment.Protein determinations were conducted on aliquots of the test materials.

Chemical Analysis

The pd-pGH was found to be only 85% pGH monomer by protein chemicalanalysis, and therefore was administered at 1.18×the dose on the basisof lyophilized material weight out. All injection solutions wereanalyzed for protein content by Bradford assay, using BSA in WHO bufferas a standard. The preparations varied in matching the expected proteinconcentrations (Table 6). In the absence of accurate methods todetermine protein concentrations of the pGH injectates by extinctioncoefficients, all actual doses were taken to be those indicated by theBradford results.

Weight Gain

The rate of weight gain was accelerated in a dose-related manner by allpreparations. Therefore, biological response was evaluated by the actualweight change from Day 0 to Day 10. The 21K pGH analog of the presentinvention induced greater weight gain than recombinant 22K pGH andpd-pGH at the lower two doses. The highest 21K pGH analog dose testeddid not stimulate weight gain further, possibly because maximal rate hadbeen achieved by lower doses. Table 7 lists the actual body weightchanges. Table 8 displays relative potencies of each preparation asdetermined by linear regression analysis of log(dose) vs. change in bodyweight, using regression coefficients for the pituitary standards.

                  TABLE 6                                                         ______________________________________                                        Protein Concentrations of                                                     Somatotropin Injectates (μg/ml)                                            Sample       Expected [ ]                                                                              Actual [ ] (Bradford)                                ______________________________________                                        pd-pGH*      353         360                                                               118         118                                                                35          35                                                  pGH (21K analog)                                                                           300         342                                                               100         127                                                                30          35                                                  pGH (21K analog)                                                                           300         396                                                               100         138                                                                30          36                                                  pit-bGH "std."                                                                             300         329                                                               100         109                                                                30          31                                                  pGH (21K analog)                                                                           300         314                                                               100         104                                                                30          29                                                  pGH (21K analog)                                                                           300         268                                                               100          90                                                                30          24                                                  ______________________________________                                         *Estimated only 85% pGH monomer by weight                                

                  TABLE 7                                                         ______________________________________                                        Based on Bradford [Protein]                                                   Pituitary Vs. Recombinant Porcine Growth Hormone                              Hypophysectomized Rat Weight Gain Assay TRL 031-017                           (change in body weight, grams mean +/- SEM)                                   DOSE                                                                          (ug/rat/                                                                             BUF-     pit-bGH std.                                                  day)   FER      (pituitary)                                                                              22K pGH  21K pGH                                   ______________________________________                                        0      1.5 +/-                                                                       0.6                                                                    7.0             14.3 +/- 0.7                                                                             13.9 +/- 1.1                                       7.2                                 20.7 +/- 1.1                              23.6            19.4 +/- 0.8                                                  25.4                       23.6 +/- 1.2                                       27.6                                28.4 +/- 1.2                              68.4                       30.2 +/- 2.2                                       72.0            30.2 +/- 1.6                                                  79.2                                29.6 +/- 3.9                              ______________________________________                                    

                  TABLE 8                                                         ______________________________________                                        Relative Potency Equivalence of                                               Growth Hormone Preparations                                                   (Mean +/- standard error of the mean)                                         Reference               Relative Equivalence to                               Preparation                                                                              Sample       Reference Preparation                                 ______________________________________                                        pit-bGH "std"                                                                            pit-bGH "std"                                                                              1.01 ± 0.05                                        pit-bGH "std"                                                                            22K pGH      1.06 ± 0.01                                        pit-bGH "std"                                                                            21K pGH      1.40 ± 0.15                                        ______________________________________                                    

While the present invention has been described in terms of a preferredembodiment, it is expected that modifications and improvements willoccur to those skilled in the art upon consideration of this disclosure.Accordingly, it is intended that the appended claims cover all suchequivalents which come within the scope of the invention as claimed.

What is claimed is:
 1. A method for promoting growth in an animalcomprising administering to an animal an effective dose of a porcinegrowth hormone analog comprising the amino acid sequence represented bythe formula

    Z-pGH.sub.1-31 -pGH.sub.39-190

wherein Z is hydrogen, MET, ALA, or MET-ALA and is essentially free ofother proteins of porcine origin.
 2. The method according to claim 1wherein Z is ALA or MET-ALA.
 3. A method for promoting growth in ananimal comprising administering to an animal an effective dose of aporcine growth hormone analog composition comprising a porcine growthhormone analog in combination with a suitable carrier wherein saidanalog is represented by the formula

    Z-pGH.sub.1-31 -pGH.sub.39-190

wherein Z is hydrogen, MET, ALA, or MET-ALA and is essentially free ofother proteins of porcine origin.
 4. The method according to claim 3wherein Z is ALA or MET-ALA.